The defect leading to type-1 diabetes is autoimmune ablation of the pancreatic β-cells, including those arising from infection, causing severely impaired glucose uptake from the circulation (Amrani et al., 2000, Nature 406:732-42; Galleri et al., 2012, Adv Exp Med Biol 771:252-71; Grieco et al., 2012, Clin Exp Immunol 168:24-9). In type-2 diabetes, the insulin signaling cascade is impaired in the insulin responsive tissues: skeletal muscle, adipose tissue and liver (Frojdo et al., 2009, Biochimica et biophysica acta 1729:83-92). The action of insulin as a metabolic regulator and a growth factor is protein tyrosine kinase (PTK)-dependent and is an essential step in the initiation of signaling cascade initiated by its cell surface receptor. The insulin receptor is a heterotetramer of two α- and two β-subunits (Lee and Pilch, 1994, Am J Physiol 266:C319-34; Ward et al., 2013, Bioessays 35:945-54). The α-subunits are entirely extracellular and contain the ligand-binding site. The β-subunits are composed of three domains: extracellular, which is disulfide bonded to the C-terminal region of the α-subunits, transmembrane and cytosolic. The cytosolic domain in include a tyrosine kinase domain (IRK) contains three tyrosine residues, Y1158,1162,1163 that are autophosphorylated upon ligand binding to the α subunits. Autophosphorylation stimulates a change in conformation of the activation loop that is necessary for binding and tyrosine phosphorylation of proteins containing YMXM sequence (Shoelson et al., 1992, PNAS 89:2027-31) including IRS1, IRS2, PI3K and BVR, and allows assembly of multi-protein signaling complexes (Lavan et al., 1997, J Biol Chem 272:21403-7; Rocchi et al., 1998, Mol Endocrinol 12:914-23; White and Yunesh, 1998, Curr Top Microbiol Immunol 228:179-208; Cai et al., 2003, J Biol Chem 278:25323-30; Grusovin and Macaulay, 2003, Front Biosci 8:d620-41; Lerner-Marmarosh et al., 2005, PNAS 102:7109-14). Defective insulin signal transduction is associated with molecular defects in the signaling pathway, the evaluation of which has largely focused on major nodes in the pathways—the insulin receptor itself, insulin receptor substrates 1 and 2, PI3K, Akt/PKB, atypical PKCs (aPKCs, i.e. ζ or λ) and MAPKs (Frojdo et al., 2009, Biochimica et biophysica acta 1729:83-92).
Activation of IRK stimulates the Akt/GSK axis, which is vital for glucose uptake and metabolism (Hernandex et al., 2001, FEBS Lett 494:225-31; Riley et al., 2006, J Biol Chem 281:6010-9). Activated IRK phosphorylates the adapter molecules IRS1 and IRS2, which in turn recruit other proteins to the complex including PI3K. Activation of PI3K results in synthesis of phosphatidylinositol-3-phosphates, which act as membrane anchors for pleckstrin homology domain-containing proteins, such as PKCs, PDK1 and Akts(1-3). Three Akt isoforms have been characterized (Jones et al., 1991, PNAS 88:4171-5; Datta et al., 1999, Genes Dev 13:2905-27); Akt1 is the most intensely studied isoform. Unlike Akt1 or Akt2, Akt3, which is highly expressed in testis and brain, appears to play no role in glucose homeostasis (Brodbeck et al., 1999, J Biol Chem 274:9133-6; Tschopp et al., 2005, Development 132:2943-54). The catalytic domain of all Akt kinases has a threonine residue in the activation loop, T308, that is phosphorylated by PDK1 after both proteins have been recruited to the membrane. A second phosphorylation, at S473 in the hydrophobic loop leads to maximal activity (Stokoe et al., 1997, Science 277:567-70; Stephens et al., 1998, Science 279:710-4). The mechanism of serine phosphorylation has been attributed to autophosphorylation and to several other kinases (Hanada et al., 2004, Biochimica et biophysica acta 1697:3-16; Hemmings and Restuccia, 2012, Cold Spring Harb Perspect Biol 4:a011189). A threonine450 in the turn motif of the C-terminal regulatory domain is also a phosphorylation target; modification of this residue, however, is not required for full activity. Activated Akt in turn regulates a wide variety of cellular functions, including that of one of its substrates, glucose synthase kinase-3 (GSK3). The GSK-3 α and β isoforms are inactivated after phosphorylation by Akt; inactivation of GSK3 stimulates glucose uptake and also allows activation of glycogen synthase, and hence synthesis of glycogen (Orena et al., 2000, J Biol Chem 275:15765-72; Buller et al., 2008, Am J Physiol Cell Physiol 294:C836-43).
Human (h)BVR is a 296 residue soluble protein that was initially described as being the sole cellular source of bilirubin, a most potent intracellular quencher of free radicals (Stocker et al., 1987, Science 235:1043-6; Stocker et al., 2004, Antioxid Redox Signal 6:841-9; Vitek, 2012, Front Pharmacol 3:55). However, hBVR is also a bZip (basic zipper) transcription factor for regulation of stress response genes, including ATF2/CREB (Kravets et al., 2004, J Biol Chem 279:19916-23; Ahmad et al., 2002, J Biol Chem 277:9226-32) and a Ser/Thr/Tyr kinase that is activated by IRK (Lerner-Marmarosh et al., 2005, PNAS 102:7109-14; Salim et al., 2001, J Biol Chem 276:10929-34) hBVR translocates, depending on the stimulus, to and from the nucleus, cytoplasm or cell membrane (Lerner-Marmarosh et al., 2007, FASEB J 21:3949-62; Lerner-Marmarosh et al., 2008, PNAS 105:6870-5; Maines et al., 2007, J Biol Chem 282:8110-22; Tudor et al., 2008, Biochem J 413:405-16) and in so doing functions as a scaffold and as an intracellular carrier protein (Kravets et al., 2004, J Biol Chem 279:19916-23; Ahmad et al., 2002, J Biol Chem 277:9226-32; Lerner-Marmarosh et al., 2008, PNAS 105:6870-5; Maines et al., 2007, J Biol Chem 282:8110-22; Gibbs et al., 2012, J Biol Chem 287:1066-79; Miralem et al., 2005, J Biol chem 280:17084-92). The structural features of the protein are of relevance to its many functions (Maines et al., 1996, Eur J Biochem 235:372-81; Whitby et al., 2002, J Mol Biol 319:1199-210; Kikuchi et al., 2001, Nat Struct biol 8:221-5); the N-terminal domain includes the active site and residues involved in ATP/NADPH binding, whereas the C-terminal domain includes a large six-stranded β-sheet followed by an α-helix that forms an ideal surface for protein: protein interaction.
Insulin was shown to regulate components of heme degradation pathway. It induced HO-1 expression through IRS1/PI3K/Akt2 pathway (Geraldes et al., 2008, J Biol Chem 283:34327-36). Also, hBVR was shown to be a substrate for the insulin receptor kinase (Lerner-Marmarosh et al., 2005, PNAS 102:7109-14), which phosphorylates three tyrosine residues in the protein: protein interactive domain in vitro. One of these, Y198, is in a canonical IRK substrate motif, YMKM, while Y228 in the YLSF motif that meets YΦSΦ criteria for an IRK target (Favelyukis et al., 2001, Nat Struct Biol 8:1058-63) and Y291 is in the C-terminal helix of hBVR (aa271-296) (Lerner-Marmarosh et al., 2005, PNAS 102:7109-14). hBVR functions have been systematically dissected using synthetic peptides based on its primary sequence, and tested the peptides in vitro and in cell culture systems; one such fragment corresponding to its C-terminal 7 residues, K291YCCRSK (hereinafter P2) is an activator of IRK, by means of a novel intracellular interaction with the kinase and stimulator of glucose uptake (Gibbs et al., 2014, FASEB J 28:2478-91). The peptide was shown to increase IRK Vmax, without changing Km of the kinase. It stimulated glucose uptake in 4 cell lines tested so far. Change in fluorescence emission spectra of IRK domain (aa 988-1263), with fluorophore coupled to cysteines, C1056, C1138 or C1234 in the presence of KYCCSRK (SEQ ID NO: 2) indicated that the peptide bound to and changed IRK conformation. Binding of the sequence to IRK was substantiated by finding that KYCCSRK (SEQ ID NO: 2) sequence in intact hBVR was necessary for the hBVR/IRK cross-activation (Gibbs et al., 2014, FASEB J 28:2478-91).
P2 stimulates glucose uptake in several cell types, including cells derived from liver (HepG2), kidney (HEK), pulmonary artery smooth muscle (PASM) and skeletal muscle myoblasts, using an N-myristoylated form of the peptide synthesized using the naturally occurring L-enantiomeric amino acids. This form was membrane permeable and effective for a brief period in the cultured cells (Gibbs et al., 2014, FASEB J 28:2478-91). For a number of reasons, this composition is unsuitable for use as a therapeutic agent; small molecules (MW <5 kDa) are rapidly depleted in the circulation and excreted via the kidneys, and moreover the L-amino acid peptides are highly susceptible to proteolytic degradation (Sato et al., 2006, Curr Opin Biotechnol 17:638-42; McGregor, 2008, Curr Opin Pharmacol 8:616-9).
Thus, there is a need in the art for compositions and methods for treating diabetes. The present invention satisfies this unmet need.